Littérature scientifique sur le sujet « Immunereconstitution »

Abstract Background: One of the major concern for ASCT in HIV-positive patients (pts) is that post-transplant immunosuppression might worsen the immunosuppression and enhance the HIV replication. Therefore we analyzed whether the immune system of HIV-positive pts might support an immune recovery similar to the HIV-negative pts who underwent the same ASCT program. Methods: The kinetics and the extent of immunereconstitution were assessed by measuring: TCR excision circles (TRECs), CD4, CD8, CD56, CD4, CD45RA, CD19 cells, and HIV pro and viraemia Results: 15 HIV-positive and 7 HIV-negative pts with relapsed DBCL underwent ASCT. Before the induction therapy TRECs/106 PBMC mean value was 962±2183 and 2948±5485 in HIV-positive and HIV-negative pts respectively. The nadir was reached before ASCT (150±11 vs 927±1842). At one year the mean value of TRECs/106 PBMC returned to the baseline in HIV-negative pts, while in HIV-positive pts largely overcame the baseline (2440±2799). Before the induction therapy, HIV-positive pts showed a significant lower CD4 (174±110 vs 386±200) CD56 (86±129 vs 144±92) and CD4/CD8 ratio in comparison with HIV-negative pts. CD4 count nadir was reached during aplastic period in both groups. No differences were present in the dynamics of CD4, CD45RA, CD8 and CD56 recovery between the two groups. At one year CD4 count returned to the baseline value in HIV-negative pts while in HIV-positive pts overcame 70% the baseline value. Before ASCT, all HIV-positive pts were on HAART and HIV-viraemia was <50cp/ml in 10/15; 6 pts discontinued HAART, but HIV viraemia and proviraemia did not increase significantly during the overall observational period. Conclusions: The dynamics of the immunereconstitution was similar in HIV-positive and HIV-negative pts, but the tymic output unexpectedly seemed to be enhanced in HIV-positive pts. Supported by ISS grants.

Abstract T-cell depletion of allogeneic hematopoietic stem cell graft (HSCT) represents the most powerful approach to prevent graft-versus-host disease (GvHD), thus allowing to overcome HLA barriers in patients with high risk malignancies, lacking a conventional donor. We hypothesized that early add-back of suicide-gene transduced donor lymphocytes (TK cells) to leukemic patients undergoing haploidentical HSCT (haplo-HSCT) could provide early immune-reconstitution and selective control of GvHD. In a phase II clinical trial (protocol MMTK007), 17 of 29 enrolled pts, (median age 52), received add-backs of 10^7/kg TK cells 42 days after haplo-HSCT. TK cells engraftment, observed in 14 patients, was necessary and sufficient for a rapid and effective immunereconstitution (IR), with a median of 144 (101–336) CD3+, 59 (28–149) CD4+ and 86 (52–279) CD8+ cells/mcl at day 100 after HSCT. Accordingly, engraftment of TK cells was tightly correlated with clinical outcome: while 3/3 pts who failed TK cells engraftment died of infections, only 1/14 TK engrafted patients died from infections (last event at day +166). As shown in Table I, the immune repertoire of treated patients improved significantly at 6 months post transplant and normalized completely in 12 months. High numbers of T cell precursors specific for CMV and EBV were detected at immune reconstitution (median of 86 and 69 gIFN specific spots/10^5 PBMC respectively) and predicted subsequent freedom from viral reactivation (p=0.002). Six pts developed acute (GvHD), (grade I to IV) that was always completely abrogated by the suicide system. Overall survival of TK cells treated patients is 50% at three years. These results indicate that TK-DLI abolish late mortality after CD34+ haplo-SCT in adults. A phase III multicentric study will start in 2007 to validate prospectively the advantage of TK-DLI in haplo-SCT.

Despite a rapidly accumulating clinical experience with autologous stem cell transplantation (ASCT) as a treatment for severe refractory autoimmune disease, data on the mechanisms by which ASCT induces immune tolerance are still very scarce. In this study it is shown that ASCT restores immunologic self-tolerance in juvenile idiopathic arthritis (JIA) via 2 mechanisms. First, ASCT induces a restoration of the frequency of FoxP3 expressing CD4+CD25bright regulatory T cells (Tregs) from severely reduced numbers before ASCT to normal levels after ASCT. This recovery is due to a preferential homeostatic expansion of CD4+CD25+ Tregs during the lymphopenic phase of immunereconstitution, as measured by Ki67 and CD44 expression, and to a renewed thymopoiesis of naive mRNA FoxP3 expressing CD4+CD25+ Tregs after ASCT. Second, using artificial antigen-presenting cells to specifically isolate self-reactive T cells, we demonstrate that ASCT induces autoimmune cells to deviate from a proinflammatory phenotype (mRNA interferon-γ [IFN-γ] and T-bet high) to a tolerant phenotype (mRNA interleukin-10 [IL-10] and GATA-3 high). These data are the first to demonstrate the qualitative immunologic changes that are responsible for the induction of immune tolerance by ASCT for JIA: the restoration of the CD4+CD25+ immune regulatory network and reprogramming of autoreactive T cells.

Abstract The outcome of haplo-SCT is limited by delayed immune reconstitution resulting in a high rate of late mortality and relapse. Here, we report results of a phase II multicenter trial (MM TK007) of early add-backs of donor lymphocytes genetically engineered to express the herpes simplex thymidine kinase (TK) suicide gene after haplo-SCT in inducing immune reconstitution and selective control of GvHD by ganciclovir. Twentysix advanced age pts (median age 51, 17–63) were transplanted for high risk leukemia; disease status at SCT was CR1 (8), CR2 (7), refractory (11). A median of 12.2x106/kg (7.3–16.8) CD34+ selected (Clinimacs) and 1x104/kg (0.8–1.4) CD3+ cells were infused after a myeloablative conditioning. 24/26 pts engrafted with a median time of 14 d (8–21) for ANC >1.0x109/l and 13 d (11–24) for plt >50x109/l. No immune reconstitution and no GvHD were observed in absence of TK-add-back. Sixteen pts received TK-DLI at a median dose of 107/kg with 1st infusion at d +42 and 13 pts obtained CD3+ >100/mcl at a median time of 91 d (61–127) from SCT and 24 d (14–42) from TK-DLI. Transduced cells were documented ex vivo in all pts and represented a median of 48% (10–90) of CD3+ cells. Five pts developed acute GvHD, (grade I to IV) that was always completely abrogated by ganciclovir. In patients in CR at time of SCT who were alive at d +42 and received add-backs of Tk cells, OS rate was 46% at 800 days (intention-to-treat analysis: 38% OS at 800 days post-SCT). Of significance, the cumulative incidence of TRM and relapse showed a 40% probability of mortality with a median time of death of 90 days and last event at day +166. This figure indicate that TK cell add-backs abolish late mortality after CD34+ SCT in adults. In patients in relapse at time of HSCT, a median OS of 201 days was obtained in ITT, with a significant advantage on expected survival without transplantation (60 d) and superior results as compared to haplo EBMT registry (80 d). The 2-year estimation of events of this multicenter phase II study confirm that TK-DLI is an effective tool for promoting immune reconstitution and protecting pts from late infectious mortality after haplo-SCT. We believe that these results are due to the rapid development of a wide T cell repertoire obtained by TK cell infusions. Immunological follow-up showed Th1/Tc1 effector memory T cells, with a wide TCR repertoire in the first 3 months after SCT in all patients. High frequencies of T cells specific for CMV (median: 35 and 93 spots/105 cells with CMV-infected donor and host fibroblasts) and EBV (median: 58 and 41 spots/105 cells with donor and host EBV-LCL) were detected by gIFN ELISpot at time of immunereconstitution, and correlated with complete control of viral infections. Normalization of the T cell repertoire was documented by spectratype, immune-phenotype for naïve and memory T cell subsets and gIFN ELIspot 6 months after treatment. A phase III randomized multicentric study will start in 2006.

Il trapianto di cellule ematopoietiche (HCT) rappresenta una terapia cardine per il trattamento delle neoplasie ematologiche altrimenti incurabili. Tuttavia, la procedura di trapianto può essere gravata dalla recidiva della malattia, dalla malattia del trapianto contro l'ospite (GVHD) e dalle infezioni. Le cellule T e NK che ricostituiscono dopo l'HCT proteggono da infezioni e recidive, ma sono anche coinvolte nella patogenesi della GVHD. Gli obiettivi del mio progetto di dottorato erano di migliorare la comprensione della ricostituzione delle cellule T e NK, utilizzando campioni di donatori sani e pazienti dopo il trapianto e diversi approcci tecnici (citometria a flusso, citometria di massa, sequenziamento dell'RNA e test funzionale ex vivo) e sviluppare nuove strategie immunoterapiche basate sui linfociti T e NK dopo HCT. In primo luogo, abbiamo dimostrato che un ritardo nel recupero dei linfociti T, un rapporto Treg/Tcon più elevato, un'aumentata espressione di PD-1 sui linfociti T di memoria e un arricchimento di cellule NK a fenotipo immaturo sono stati osservati dopo HCT aploidentico (aplo-HCT) con l’utilizzo di ciclofosfamide post-trapianto. Inoltre, la funzione delle cellule NK CD56brightCD16+ immature funzionalmente alterate dopo aplo-HCT può essere migliorata con l’utilizzo dell'interleuchina-15 in vitro. In secondo luogo, abbiamo avviato uno studio di fase I sulle cellule cytokine-induced memory-like (CIML) NK infuse da donatore haploidentico in pazienti con neoplasie mieloidi che hanno avuto una recidiva dopo aplo-HCT. Nei primi 6 pazienti arruolati, l'infusione di cellule CIML-NK ha portato a una rapida espansione in vivo da 10 a 50 volte, che è stata mantenuta per mesi. L'infusione è stata ben tollerata, con febbre e pancitopenia come eventi avversi più comuni. Sulla base di questi dati preliminari, le cellule CIML-NK possono fungere da piattaforma per il trattamento della recidiva post-trapianto delle patologie mieloidi. Infine, ci siamo concentrati sul bilanciamento della risposta dei linfociti T per controllare l’incidenza di GVHD. CD6, un recettore co-stimolatorio dei linfociti T, che aiuta a stabilizzare la sinapsi immunologica tra la cellula T e l'APC, dopo legame con il suo ligando, la molecola di adesione delle cellule leucocitarie attivate (ALCAM). In questo contesto, il legame CD6-ALCAM promuove l'attivazione, la proliferazione e la maturazione delle cellule T. Abbiamo dimostrato che le cellule T CD6 ricostituivano subito dopo il trapianto, con le cellule Treg che esprimono livelli inferiori di CD6 rispetto alle cellule Tcon e cellule T CD8+. Dopo l'insorgenza della aGVHD, l'espressione sia di CD6 che di ALCAM è stata mantenuta. Itolizumab ha inibito l'attivazione e la proliferazione delle cellule T CD4+ e CD8+ nell'ambito di aGVHD in esperimenti ex vivo, senza mediare l'attività citolitica diretta o la citotossicità anticorpo-dipendente. I nostri risultati identificano la via di CD6-ALCAM come potenziale bersaglio per il controllo dell'aGVHD. Uno studio di fase I/II che utilizza itolizumab come trattamento di prima linea in combinazione con steroidi per i pazienti con aGVHD è attualmente in corso. In conclusione, questi risultati evidenziano la necessità di bilanciare le proprietà effettrici e tolerogeniche del sistema immunitario che si ricostituisce dopo HCT e suggeriscono differenti strategie per promuovere o moderare le funzioni delle cellule T e NK.
Hematopoietic cell transplantation (HCT) represents a cardinal therapy for hematological malignancy otherwise incurable. However, HCT can be complicated by disease recurrence, graft versus host disease (GVHD) and infections. After HCT, reconstituting T and NK cells protect against infection and relapse, but they are also involved in the pathogenesis of GVHD. The aims of my PhD project were to improve the understanding of T and NK-cell reconstitution, using samples from both healthy donor and patients after transplant and different technical approaches (flow cytometry, mass cytometry, RNA sequencing, and ex vivo functional assay) and to develop post-transplant T and NK cell-based immunotherapeutic strategies. First, we showed that delayed early T-cell recovery, a higher Treg/ Tcon ratio, an increased PD-1 expression on memory T cells, and an enriched immature NK phenotype were observed after haploidentical HCT (haplo-HCT) with post-transplant cyclophosphamide. In addition, the expansion of functionally impaired immature CD56brightCD16+ NK cells after haplo-HCT can be enhanced with in vitro interleukin-15 priming. Second, we initiated a phase I trial of adoptively transferred cytokine-induced memory-like (CIML) NK cells in patients with myeloid malignancies who relapsed after haplo-HCT. In the first 6 enrolled patients, infusion of CIML NK cells led to a rapid 10- to 50-fold in vivo expansion that was sustained over months. The infusion was well tolerated, with fever and pancytopenia as the most common adverse events. Based on these preliminary data, CIML NK cells may serve as a promising platform for the treatment of posttransplant relapse of myeloid disease. Finally, we focused on the balancing of T cell response to control GVHD occurrence. CD6, a pan-T cell co-stimulatory receptor, helps to stabilize the immunological synapse between the T cell and the APC, upon ligation, with its ligand, activated leukocyte cell adhesion molecule (ALCAM). In this context, CD6-ALCAM binding promotes T cell activation, proliferation, maturation. We showed that CD6 T cells reconstituted early after transplant with Treg expressing lower levels of CD6 compared to Tcon and CD8+ T cells. After onset of aGVHD, both CD6 and ALCAM expression was maintained. Itolizumab inhibited CD4+ and CD8+ T cell activation and proliferation in the setting of aGVHD in ex vivo experiments, without mediate direct cytolytic activity or antibody-dependent cytotoxicity. Our results identify the CD6-ALCAM pathway as a potential target for aGVHD control. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing. In conclusion, these results highlight the need of balancing the effector and tolerogenic properties of the immune system reconstituting after HCT and suggest different strategies to enhance or moderate the T and NK cells functions.

Human leukocyte antigen (HLA)-mismatched haematopoietic stem cell transplantation (HSCT) represents an important therapeutic option for patients lacking suitable donors. Delayed posttransplant immune recovery constitutes one of its major complications and is most pronounced in the T cellular compartment. A novel strategy to promote de novo thymopoiesis from donor derived HSCs and to accelerate T cellular reconstitution in patients after HSCT consists in the adoptive transfer of in vitro generated T cell progenitor cells. Identification of Notch1 as the key regulator of early T-lineage development has allowed the generation of Notch ligand-based culture systems, which provide a powerful tool to generate T-lymphoid progenitors in vitro. The efficacy of murine T-lymphoid progenitors to promote T cell reconstitution has been well demonstrated in conventional mouse models. In consistency, in vitro-generated human T cell progenitors were demonstrated to promote thymic recovery in humanized mice. Yet, positive effects of in vitro generated human T cell precursors on peripheral T cell reconstitution have not been demonstrated. Moreover currently used Notch-based co-culture systems consist of genetically modified murine cell lines. With view to establishing a clinically applicable system, feeder-cell-free Notch-ligand culture systems for the generation of T-lymphopoietic progenitors are warranted. During my PhD project I developed a new culture system based on the immobilized Notch ligand Delta-like-4 (DL-4). Exposure of human CD34+ cord blood cells to immobilized DL-4 enabled the in vitro generation of high number of T cell progenitors, which harboured the phenotype of immature early thymic progenitor cells (ETP) and prothymocytes (proT). ETP and proT cell generated during DL-4 culture upregulated essential genes involved in early T-lymphoid development (i.e. IL7Rα, PTα, RAG1 and BCL11b) and had undergone stage-specific recombination of the T cell receptor (TCR) locus in a similar way as in native human thymopoiesis. In limiting dilution analysis after secondary OP9/DL-1 co-culture, DL-4 progenitors displayed a highly increased T-lymphoid potential, which could be entirely attributed to the ETP and proT subset. When transferred into NOD/SCID/γc-/- mice, DL-4 primed T cell progenitors migrated to the thymus and accelerated intrathymic T cell differentiation and emergence of functional, mature and polyclonal αβ T cells in the periphery. In a co-transplantation approach, which more closely mimics a clinical setting, DL-4 progenitors and untreated CD34+ cells from HLA-disparate donors were simultaneously injected in the same recipient. This procedure allowed even more rapid and more robust T cell reconstitution. HLA-tracking of the distinct graft sources further showed, that DL-4 progenitors specifically reconstituted the T-lymphoid compartments. This work provides further evidence for the ability of in vitro-generated human T cell progenitors to promote de novo thymopoiesis and shows for the first time, that these cells accelerate peripheral T cell reconstitution in humanized mice. The availability of the efficient feeder-cell-free DL-4 culture technique represents an important step towards the future clinical exploitation translation of in vitro generated T-lymphoid progenitor cells to improve posttransplant immune reconstitution